One of the most difficult steps in the study of biological macromolecules is he isolation of sufficient amounts of pure species with retention of native configuration. Reverse phase high pressure liquid chromatography in both conventional and narrow bore format and electrophoresis in SDS buffer systems followed by electroelution or electroblotting of resolved components represented the most efficient methods whenever affinity or other gel chromatography techniques are not applicable. However, many macromolecules of interest are not enable to these approaches due to low recovery and loss of biological activity ring separation in the presence of organic solvents or sodium dodecyl sulfate. We are developing techniques to purify biologically active molecules by multiphasic zone electrophoresis in polyacrylamide gel under non-denaturing conditions, in conjunction with HPEC and elution under carefully controlled temperature and buffer composition. The buffer systems are designed to encompass operative pH's ranging from 7.5 to 11.0 and to provide enough mobility for proteins with acidic to basic isoelectric points. The high resolving power of polyacrylamide gel electrophoresis and the ability to separate molecules on the basis of differences in size, net charge and hydrophobicity will be applied to the isolation of native cytokines, human goiter proteins and tetanus toxins.